Kinetic analysis of N-alkylaryl carboxamide hexitol nucleotides as substrates for evolved polymerases

Abstract

Six 1',5'-anhydrohexitol uridine triphosphates were synthesized with aromatic substitutions appended via a carboxamide linker to the 5-position of their bases. An improved method for obtaining such 5-substituted hexitol nucleosides and nucleotides is described. The incorporation profile of the nucleotide analogues into a DNA duplex overhang using recently evolved XNA polymerases is compared. Long, mixed HNA sequences featuring the base modifications are generated. The apparent binding affinity of four of the nucleotides to the enzyme, the rate of the chemical step and of product release, plus the specificity constant for the incorporation of these modified nucleotides into a DNA duplex overhang using the HNA polymerase T6G12_I521L are determined via pre-steady-state kinetics. HNA polymers displaying aromatic functional groups could have significant impact on the isolation of stable and high-affinity binders and catalysts, or on the design of nanomaterials.

Figure 1.

(A) Schematic presentation of the modified nucleotides used in this study, 1a–f, (B) the aryl (R) moieties in 1a–f.

Scheme 1.

Synthesis of the 5-substituted hUTP nucleotides 1a–f; (i) 5-iodouracil, DBU, CH₃CN, 80°C, 16 h, 63%; (ii) N³-benzoyliodouracil, PPh₃, DEAD, dioxane, 18 h, RT, 75%; (iii) NH₃, MeOH, 90 min, RT, 65%; (iv) Mo(CO)₆, NEt₄Cl, Bu₃N, RNH₂, diglyme, 100–120°C, 2 h, 65–92%; (v) 1,1′-Thiocarbonyldiimidazole, dichloromethane, reflux, 8 h, then Bu₃SnH, AIBN, toluene, 1–1.5 h; 80–82%; (vi) 80% AcOH, 40°C, 1 h, 30–60%; (vii) POCl₃, TMP, Proton-Sponge, 0°C, 5 h; (viii) Bu₃N, (NBu₄)₃HP₂O₇, DMF, 0–25°C, 30 min, 30–40%; (ix) Pd, cyclohexene.

Figure 2.

The incorporation of hTTP (H) and the 5-substituted hUTPs 1a-f (lanes a–f) opposite a poly-dA template overhang in a DNA duplex using the evolved HNA polymerases T6G12_I521L and T6G12. The modified nucleotides (hTTP and 1a–f) are used at a concentration of 125 μM. After optimization of the reaction conditions, the enzymes are used at a final concentration of 51 nM for T6G12_I521L and 82 nM T6G12. The reactions with the enzyme T6G12 contain 0.5 mM freshly prepared MnCl₂. All reactions are carried out in 1× Thermopol buffer (NEB) supplemented with 1.5 mM MgSO₄. The reactions are incubated at 50°C overnight. The positions where a 5-substituted hUTP has to be incorporated opposite the template oligonucleotide, are underlined in the sequence below the gel image. The lanes indicated with ‘P’ show the primer control (no enzyme and no nucleotides added). The position for the full-length material of HNA (signifying the incorporation of ten hT nucleotides) is indicated by ‘FL HNA’ on the side of the gel image.

Figure 3.

The incorporation of the 5-substituted hUTPs together with hATP, hCTP and hGTP into the P1T2 duplex overhang using T6G12_I 521L after cycling for 1 min at 94°C, followed by 5 min at 50°C and 2 h 65°C, for 16 h in total, in Thermopol buffer 1× containing an additional 2 mM MgSO₄. ‘P’ indicates the primer control. Lane H shows the hNTP control. Lanes a + hACG to f + hACG show the incorporation of the hA, hC and hG building blocks together with 1a–f respectively.

Figure 4.

The burst kinetic profile of the incorporation of hTTP at a concentration of 75 μM into the growing duplex P2T3 (100 nM) using T6G12_I521L at a concentration of 14.9 nM as determined via the titration of the enzyme active site. Graphpad Prism v6 was used for data fitting as described in the Materials and Methods section (R² value of 0.997).